Transferencia del material
genético.
Conjugación, transformación y
transducción.
Mapeo genético
Mutations in Bacteria
• Mutations arise in bacterial populations
– Induced
– Spontaneous
• Rare mutations are expressed
– Bacteria are haploid
– Rapid growth rate
• Selective advantage enriches for mutants
• Gene transfer occurs in bacteria
Gene Mapping in Bacteria and
Bacteriophages
Mapping bacteria, 3 different methods:
 Conjugation
 Transformation
 Transduction
Bacteriophage mapping:
 Bacteriophage gene mapping
 Cis-trans complementation test
Bacteria transfer (or receive) genetic
material 3 different ways
Transfer always is unidirectional, and
no complete diploid stage forms.
1. Conjugation
2. Transformation
3. Transduction
Mating types in bacteria
– Donor
• F factor (Fertility factor)
– F (sex) pilus
– Recipient
Donor
• Lacks an F factor
Recipient
General Features of
Gene Transfer in Bacteria
• Unidirectional
– Donor to recipient
• Donor does not give an entire
chromosome
– Merozygotes
• Gene transfer can occur between
species
Conjugation
1. Discovered by Joshua Lederberg and Edward
Tatum in 1946.
2. Unidirectional transfer of genetic material
between donor and recipient cells by direct
contact.
3. Segment (rarely all) of the donor’s
chromosome recombines with the
homologous recipient chromosome.
4. Recipients containing donor DNA are called
transconjugants.
Lederberg & Tatum
(1946) Experiment
demonstrating
recombination in E.
coli.
• Recombination
of 2
complimentary
auxotrophs
gives rise to a
strain that can
synthesize all
nutrients.
Bernard Davis experiment demonstrated that
physical contact is required for bacterial
recombination.
E. coli conjugation
Conjugation-transfer of the
sex factor F
1. William Hayes (1953) demonstrated that
genetic exchange in E. coli occurs in only
one direction.
2. Genetic transfer is mediated by sex factor F.
3. Donor is F+ and recipient is F-.
4. F is a self-replicating, circular DNA plasmid
(1/40 the size of the main chromosome).
Conjugation-transfer of the
sex factor F
5. F plasmid contains an origin sequence (O),
which initiates DNA transfer. Also contains
genes for hair-like cell surface (F-pili or sexpili), which aid in contact between cells.
6. No conjugation can occur between cells of the
same mating type.
7. Conjugation begins when the F plasmid is
nicked at the origin, and a single strand is
transferred using the rolling circle mechanism.
8. When transfer is complete, both cells are F+
double-stranded.
Transfer of the
F factor
Conjugation of highfrequency recombinant
strains
1. No chromosomal DNA is transferred by
standard sex factor F.
2. Transfer of chromosome DNA is facilitated
by special strains of F+ integrated into the
bacteria chromosome by crossing over.
3. Hfr strains = high frequency recombination
strains.
4. Discovered by William Hayes and Luca
Cavalli-Sforza.
5. Hfr strains replicate F factor as part of their main
chromosome.
Conjugation of highfrequency recombinant
strains
5. Conjugation in Hfr strains begins when F+ is
nicked at the origin, and F+ and bacteria
chromosomal DNA are transferred using the
rolling circle mechanism.
6. Complete F+ sequence (or complete
chromosomal DNA) is rarely transferred
(1/10,000) because bacteria separate
randomly before DNA synthesis completes.
7. Recombinants are produced by crossover of
the recipient chromosome and donor DNA
containing F+.
Transfer of the
Hfr F+ factor
Excision of the F+ factor
also occurs
spontaneously at low
frequency.
1. Begin with Hfr cell
containing F+.
2. Small section of host
chromosome also may
be excised, creating an
F’ plasmid.
3. F’ plasmid is named for
the gene it carries, e.g.,
F’ (lac)
Using conjugation to map bacterial
genes
1. Begin with appropriate Hfr strains selected from
F+ x F- crosses and perform an interrupted
mating experiment.
2. HfrH
F-
thr+
leu+ aziR tonR lac+ gal+ strR
thr
leu aziS tons lac gal
strS
3. Mix 2 cell types in medium at 37°C.
Using conjugation to map bacterial
genes
4.
Remove at experimental time points and agitate
to separate conjugating pairs.
5.
Analyze recombinants with selective media.
6. Order in which genes are transferred reflects
linear sequence on chromosomes and time in
media.
7.
Frequency of recombinants declines as donor
gene enters recipient later.
Interrupted
mating
experiment
Genetic map-results of interrupted E.
coli mating experiment.
Generating a map
for all of E. coli
1. Location and orientation
of the Hfr F+ in the
circular chromosome
varies from strain to
strain.
2. Overlap in transfer
maps from different
strains allow generation
of a complete
chromosomal map.
Circular
genetic map of
E. coli
Total map units =
100 minutes
~time required for
E. coli
chromosome to
replicate at
37°C.
Significance
• Gram - bacteria
– Antibiotic resistance
– Rapid spread
• Gram + bacteria
– Production of adhesive material by
donor cells
Transformation
• Unidirectional transfer of extracellular DNA
into cells, resulting in a phenotypic change in
the recipient.
• First discovered by Frederick Griffith (1928).
• DNA from a donor bacteria is extracted and
purified, broken into fragments, and added to
a recipient strain.
• Donor and recipient have different
phenotypes and genotypes.
• If recombination occurs, new recombinant
phenotypes appear.
More about transformation
• Bacteria vary in their ability to take up DNA.
• Bacteria such as Bacillus subtilis take up DNA
naturally.
• Other strains are engineered (i.e., competent
cells).
• Competent cells are electroporated or treated
chemically to induce E. coli to take up
extracellular DNA.
Bacteria known to be capable
of transformation
• Natural transformation
– Gram positive bacteria
• Streptococcus pneumoniae, S. sanguis, B. Subtilis, B.
Cereus, B. Stearothermophilus
– Gram negative bacteria
• Neisseria gnonorrheae, Acinetobacter calcoaceticus,
Moraxella osloensis, M. urethans
• Psychrobacter sp., Azotobacter agilis, Haemophilus
influenzae, H. Parainfluenzae, Pseudomonas stutzeri
• Artificial transformation
• Escherichia coli, Salmonella thyphimurium,
Pseudomonas aeruginosas y muchas otras.
Transformation
• Steps
– Uptake of DNA
• Gram +
• Gram -
– Recombination
• Legitimate,
homologous or
general
• recA, recB and recC
genes
• Significance
Phase variation in Neiseseria
– Recombinant DNA technology
–
Transformation of Bacillus
subtilis
Heteroduplex DNA
Chemical competence
•
•
In some bacteria, including E. coli, treatment of cells with
divalent cations at low temperature, facilitates the uptake of
plasmid DNA into the cell (linear DNA can be taken up, but
is shredded by cytoplasmic DNases before it can do
anything)
Remains unclear how this works
Uptake channels
made of polyP, PHB,
and Ca
Hanahan and Bloom, 1996, Chapter 132, Escherichia coli
and Salmonella, ASM Press
Electroporation
High field strengths result in very transient holes in the cellular
envelope
Under the appropriate conditions, DNA leaks in and DNA leaks out.
A high concentration of plasmid outside results in a rapid influx of
plasmids into the cell.
Electroporation
cuvette
Cells go here
High voltage
shock
How well has your
transformation worked?
Transformation efficiency
Saturating cells (# of transformants/mg of DNA)
106-109/mg of pBR322
app. 1011 plasmids/mg pBR322
can also be analyzed as % of cells that receive plasmid
Saturating DNA
% of DNA molecules that successfully transform cells
Protocol
Sat. cells
Sat. DNA
Chemical
1%
12%
Electro
10%
90%
Natural
transformation in
Gram positives
Examples:
Streptococcus pneumoniae
Bacillus subtilis
• no base specificity
• limited # of uptake sites
(30-75)
• nicked internally
• complement is
degraded during
transport
• recombines in recipient
Dubnau. 1999. Ann. Rev. Microbiol. 53:217
Natural
transformation in
Gram negatives
Examples:
Haemophilus influenzae
Neisseriae gonorrhoeae
• sequence specific –
uptake sequences
• 4-8 sites/cell
• no cell bound intermediate
• import of ds DNA to
periplasm
• complement is degraded
during transport into
cytoplasm
• recombines in recipient
Dubnau. 1999. Ann. Rev. Microbiol. 53:217
Gram positive uptake
machinery
-dedicated machinery for the transport of DNA into the cell
the reverse of a conjugal transfer system
- some components similar to Tra functions
Dubnau. 1999. Ann. Rev. Microbiol. 53:217
Gram-negative uptake
machinary
-dedicated machinery for the transport of DNA into the cell
- must cross periplasm and outer membrane
Dubnau. 1999. Ann. Rev. Microbiol. 53:217
Energy for driving the
process?
• Intracellular ATP
hydrolysis
• pH gradient – PMF?
• Complement degradation
Function for natural
transformation
• Nutrition
• DNA repair
• Genetic diversification
Diferencias entre los sistemas de transformaci—nnatural codificados por
Streptococcus pneumoniae y Haemophilus influenzae.
Propiedad
Streptococcu s
Haemophilus
Factores de
competencia
desencadenan la competencia
S’
No
Forma en que el DNA entr a en
la cЋlula
Hebra sencilla
Hebra doble
Fuente de DNA que puede
entrar a la cЋlula
Cualquiera
S—lo hom—loga
Forma del DNA unido a l a
superficie celular
Hebra doble
Hebra doble
Estado f’sico del DNA dent ro
de la cЋlula
Unido a prote’na s
Contenido en el
transformasoma
Mapping using transformation
Recombination frequencies are used to infer
gene order.
p+q+ o+
x
p
q
o
1. If p+ and q+ frequently cotransform, order is
p-q-o.
2. If p+ and o+ frequently cotransform, order is
p-o-q.
Transduction
1.
Bacteriophages (bacterial viruses) transfer genes
to bacteria (e.g., T2, T4, T5, T6, T7, and ).
1. Generalized transduction transfers any gene.
1. Specialized transduction transfers specific
genes.
2.
Phages typically carry small amounts of DNA,
~1% of the host chromosome.
3.
Viral DNA undergoes recombination with
homologous host chromosome DNA.
Transduction
• Genetic exchange mediated
by bacterial viruses
(bacteriophage)
• Two basic types of bacterial
viruses
• Lytic viruses – infect
cells, multiply rapidly,
lyse cells
• Lysogenic viruses – infect
cells, can integrate into
genome and go
dormant
(a prophage)
•
- at some point, can
excise, multiply and
lyse cells.
Phage Composition and
Structure
• Composition
– Nucleic acid
Head/Capsid
• Genome size
• Modified bases
– Protein
• Protection
• Infection
• Structure (T4)
– Size
– Head or
capsid
Contractile
Sheath
Tail
Tail Fibers
Base Plate
Infection of Host Cells by Phages
• Adsorption
– LPS for T4
•
•
•
•
Irreversible attachment
Sheath Contraction
Nucleic acid injection
DNA uptake
Types of Bacteriophage
• Lytic or virulent – Phage that multiply within the host
cell, lyse the cell and release progeny phage (e.g.
T4)
• Lysogenic or temperate phage: Phage that can
either multiply via the lytic cycle or enter a quiescent
state in the bacterial cell. (e.g., )
– Expression of most phage genes repressed
– Prophage
– Lysogen
Bacteriophage have a range of morphologies
from simple filaments to large complex
structures
• May contain either RNA or DNA associated with a protein coat
• Almost all bacteria have phage associated with them
Brock Biology of Microorganisms, vol. 9, Chapter 8
Transfer their nucleic acid
into the host cell
Attach to specific receptors on the surface of their host
bacteria
Smithsonian (Oct 2000)
T4 bacteriophage on the surface of an E. coli cell
Life cycle
of phage 
Generalized
transduction
of E. coli by
phage P1
Transduction mapping is similar to
transformation mapping
 Gene order is determined by frequency of
recombinants.
 If recombination rate is high, genes are
close together.
 If recombination rate is low, genes are far
apart.
Mapping genes of bacteriophages
1.
Infect bacteria with phages of different genotypes using two-, three-,
or four-gene crosses  crossover.
2.
Count recombinant phage phenotypes by determining differences in
cleared areas (no bacteria growth) on a bacterial lawn.
3.
Different phage genes induce different types of clearing (small/large
clearings with fuzzy/distinct borders).
Fine structure gene-mapping of
bacteriophages
Same principles of intergenic mapping also can
be used to map mutation sites within the
same gene, intragenic mapping.
1. First evidence that the gene is sub-divisible
came from C. P. Oliver ‘s (~1940) work on
Drosophila.
2. Seymour Benzer’s (1950-60s) study of the rII
region of bacteriophage T4.
Seymour Benzer’s (1950-60s) study
of the rII region of T4
1. Studied 60 independently isolated rII mutants
crossed in all possible combinations.
2. Began with two types of traits: plaque
morphology and host range property.
1. Growth in permissive host E. coli B; all four
phage types grow.
1. Growth in non-permissive host E. coli K12();
rare r+ recombinants grow (rare because the
mutations are close to each other and
crossover is infrequent).
Seymour Benzer’s (1950-60s) study
of the rII region of T4
3.
Benzer also studied 3000 rII mutants showing
nucleotide deletions at different levels of
subdivision (nested analyses).
4.
Was able to map to T4 to level equivalent to 3
bp.
5.
Ultimately determined that the rII region is subdivisible into >300 mutable sites by series of
nested analyses and comparisons.
Benzer’s method for identifying recombinants of two
rII mutants of T4.
Benzer’s map of the rII region generated from
crosses of 60 different mutant T4 strains.
Benzer’s deletion
analysis of the rII
region of T4
No recombinants can be
produced
if
mutant
strain lacks the region
containing the mutation.
Benzer’s deletion map divided the rII region into 47
segments.
Benzer’s composite map of the rII region indicating >300
mutable sites on two different genes.
Small squares indicate point mutations mapping to a given site.
Seymour Benzer’s cis-trans
complementation test
1.
Used to determine the number of functional
units (genes) defined by a given set of
mutations, and whether two mutations occur
on the same unit or different units.
2.
If two mutants carrying a mutation of
different genes combine to create a wild type
function, two mutations compliment.
3.
If two mutants carrying a mutation of the
same gene create a mutant phenotype,
mutations do not compliment.
Seymour Benzer’s cis-trans
complementation test.
Example of complementation in
Drosophila
Transposable Genetic Elements
• Definition: Segments of DNA that are able to
move from one location to another
• Properties
– “Random” movement
– Not capable of self replication
– Transposition mediated by site-specific recombination
• Transposase
– Transposition may be accompanied by duplication
Types of Transposable
Genetic Elements
• Insertion sequences (IS)
– Definition: Elements that carry no other genes
except those involved in transposition
– Nomenclature - IS1
– Structure
– Importance
GFEDCBA
ABCDEFG
Transposase
– Mutation
– Plasmid insertion
– Phase variation
Phase Variation in
Salmonella H Antigens
H1 gene
H1
flagella
IS
H2 gene
H2
flagella
Types of Transposable Genetic
Elements
• Transposons (Tn)
– Definition: Elements that carry other genes
except those involved in transposition
– Nomenclature - Tn10
– Structure
• Composite Tns
– Importance
• Antibiotic resistance
IS
Resistance Gene(s)
IS
IS
Resistance Gene(s)
IS
Plasmids
• Definition: Extrachromosomal genetic
elements that are capable of
autonomous replication (replicon)
• Episome - a plasmid that can integrate
into the chromosome
Classification of Plasmids
• Transfer properties
– Conjugative
– Nonconjugative
• Phenotypic effects
– Fertility
– Bacteriocinogenic plasmid
– Resistance plasmid (R factors)
Structure of R Factors
• RTF
RTF
– Conjugative
plasmid
– Transfer genes
• R determinant
– Resistance
genes
– Transposons
R determinant